Chemokines are secreted pro-inflammatory proteins of small dimensions (70-130 amino acids) mostly involved in the directional migration and activation of cells, especially the extravasation of leukocytes from the blood to tissue localizations needing the recruitment of these cells (Baggiolini M et al., 1997; Rossi D and Zlotnik A, 2000; Fernandez E J and Lolis E, 2002). Usually chemokines are produced at the site of an injury, inflammation, or other tissue alteration in a paracrine or autocrine fashion, triggering cell-type specific migration and activation.
Depending on the number and the position of the conserved cysteines in the sequence, chemokines are classified into C, C—C, C—X—C and C—X3—C chemokines. Inside each of these families, chemokines can be further grouped according to the sequence homology of the entire sequence and/or specific activities. Many C—X—C chemokines such as interleukin-8 (IL-8) are chemotactic for neutrophils, while C—C chemokines are active on a variety of leukocytes including monocytes, lymphocytes, eosinophils, basophils, NK cells and dendritic cells.
A series of heptahelical, G-protein coupled, membrane receptors are the binding partners that allow chemokines to exert their biological activity on the target cells, which present specific combinations of receptors according to their state and/or type. An unified nomenclature for chemokine ligands and receptors, which were originally named by the scientists discovering them in a very heterogeneous manner, has been proposed to associate each of these molecule to a systemic name including a progressive number: CCL1, CCL2, etc. for C—C chemokines; CCR1, CCR2, etc. for C—C chemokines receptors, and so on.
The physiological effects of chemokines result from a complex and integrated system of concurrent interactions. The receptors often have overlapping ligand specificity, so that a single receptor can bind different chemokines, as well a single chemokine can bind different receptors.
Even though there are potential drawbacks in using chemokines as therapeutic agents (tendency to aggregate, promiscuous binding), these molecules offer the possibility for therapeutic intervention in pathological conditions associated to such processes, in particular by inhibiting/antagonizing specific chemokines and their receptors at the scope to preventing the excessive recruitment and activation of cells, in particular leukocytes, for a variety of indications related to inflammatory and autoimmune diseases, cancers, and bacterial or viral infections (Baggiolini M, 2001; Godessart N and Kunkel S L, 2001; Proudfoot A et al., 2000).
In particular, the N-terminal domain of chemokines is involved in receptor binding and N-terminal domain processing can either activate chemokines or render chemokines completely inactive. Amino-terminal variants of synthetic C—C chemokines have been tested for their activity as inhibitors or antagonists of the naturally occurring forms. MCP-1, MCP-3 and RANTES missing up to 8 or 9 N-terminal amino acids are inactive on monocytes and are useful as receptor antagonists in the therapy and/or in diagnosis of the diseases, in which an antagonistic activity of the chemokine effects is required (Gong J H et al., 1995; Gong J H et al., 1996; WO 99/16877). Alternatively, extension of RANTES with a methionine results in almost complete inactivation of the molecule, called Met-RANTES, which behaves as an antagonist for the authentic one (Proudfoot A E et al., 1996).
Even if the chemoattractant activity of RANTES and of CC chemokines in general has been studied mainly in connection with the specific cell membrane receptors, RANTES can interact also with Glycosaminoglycans (GAGs), highly variable, branched sugar groups added post-translationally to several proteins, generically called proteoglycans (PGs). Such proteins are present on cell membrane, in the extracellular matrix and in the blood steam, where isolated GAGs can also be present.
The interaction with GAGs is a feature common to many cell-signaling soluble molecules (interleukins, growth factors). PGs, or isolated GAGs, can form a complex with soluble molecules, probably at the scope to protect this molecule from proteolysis in the extracellular environment. It has been also proposed that GAGs may help the correct presentation of cell signaling molecules to their specific receptor and, eventually, also the modulation of target cell activation.
In the case of chemokines, the concentration into immobilized gradients at the site of inflammation and, consequently, the interaction with cell receptors and their activation state seem to be modulated by the different forms of GAGs (Hoogewerf A J et al., 1997). Therefore, it has been suggested that the modulation of such interactions may represent a therapeutic approach in inflammation and other diseases (Schwarz M K and Wells T N, 1999).
The structural requirements and functional effects of GAG-RANTES interaction have been studied in various models. RANTES binds GAGs on human umbilical vein endothelial cells (HUVECs) at micromolar concentrations with an affinity and a specificity higher then other chemokines, like MCP-1, IL-8, or MIP-1alpha. Such interaction appears to be not simply electrostatic but also depending by other parameters like length and N- and O-sulphation of the GAGs (Kuschert G S et al., 1999). GAG-defective cell lines still can bind chemokines but the presence of cell surface GAGs greatly enhances their activity on the receptors when they are at low concentrations (Ali S et al., 2000).
RANTES contains a cationic sequence composed of a dibasic site, separated by a residue to another basic residue (RKNR) at residues 44-47, which is conserved in other chemokines, like MIP-1alpha (Koopmann W and Krangel M S, 1997) and MIP-1beta (Koopmann W et al., 1999). Human RANTES variants containing single mutations in this cationic sequence have been disclosed as RANTES antagonists having potential therapeutic applications in the treatment of HIV infection and inflammatory or allergic diseases (WO 99/33989). In particular, a triple mutant of RANTES, in which three residues at positions 44, 45 and 47 have been substituted with Alanine, has lost the GAG-binding ability and it is useful in the treatment of multiple sclerosis and/or other demyelinating diseases (WO 02/28419).
Several peptides and proteins, which have become commercialized drug products, lack oral efficacy and therefore have always been administered by parenteral route. Injections are generally performed by the physician or by the medical professional staff and the patients are expected to visit a surgery or a hospital regularly in order to receive treatment. Besides the discomfort created, the time taken up by this type of application often leads to unsatisfactory compliance by the patient, particularly when the treatment extends over several months.